Homogeneous enzyme immunoassay for keratinized structures

ABSTRACT

This disclosure relates to methods and kits for determining the presence and/or amount of one or more analytes in a keratinized structure (e.g., hair) sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.Nos. 62/889,870, filed on Aug. 21, 2019, and 62/887,191, filed Aug. 15,2019, the contents of which are hereby incorporated by reference intheir entireties.

TECHNICAL FIELD

This disclosure relates to methods and kits for determining the presenceand/or amount of one or more analytes in a keratinized structure (e.g.,hair) sample and more particularly to methods and kits for doing thesame using competitive immunoassays.

BACKGROUND

Immunoassays such as radioimmunoassays (RIA) and homogeneous enzymeimmunoassays (HEIA) are useful methods for determining the presence,identity, and amount of one or more analytes of interest in a sample. Inparticular, the measurement of trace amounts of analytes, particularlychemical substances, has become essential for many health careapplications in pharmaceutical studies, therapeutic drug monitoring, andfor drug abuse detection. Often, a sample, particularly a sample of akeratinized structure (e.g., hair) or a biological fluid (e.g., oralfluid, blood, urine) obtained from an individual, is screened for thepresence of one or more analytes (e.g., illicit and non-illicit drugs).

The present disclosure relates to methods and assay that overcome thecurrent obstacles and limitations and provides homogeneous enzymeimmunoassay methods and kits for the qualitative determination of lowconcentration analytes in keratinized structure (e.g., hair) samples.

SUMMARY

Provided herein is a method for determining the presence or absence ofan analyte of interest in a keratinized structure sample comprising:combining in an aqueous medium: a conjugate comprising an enzyme and theanalyte of interest; an antibody specific for the analyte of interest;and the keratinized structure sample; and determining if the analyte ofinterest is present or absent in the keratinized structure sample.

In some embodiments, the enzyme is selected from the group consisting ofglucose-6-phosphate dehydrogenase (G6PDH), alcohol dehydrogenase,glutamic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase,α-glycerol phosphate dehydrogenase, lactic dehydrogenase,glyceraldehydes-3-phosphate dehydrogenase, gluthathione reductase,quinine reductase, nitrate reductase, and glutamic dehydrogenase. Insome embodiments, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH). In some embodiments, the G6PDH is covalently linked to theanalyte of interest.

In some embodiments, the antibody specific for the analyte of interestis selected from the group consisting of cocaine, benzoylecgonine,cocaethylene, norcocaine, PCP, amphetamine, methamphetamine,cannabinoids, THC, carboxy-THC, heroin, benzodiazepines, methadone,codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA). In some embodiments, theantibody is detectably labeled.

In some embodiments, the G6PDH is obtained from a natural source. Insome embodiments, the G6PDH is a recombinant enzyme.

In some embodiments, the aqueous medium further comprises: an enzymesubstrate for G6PDH; and a coenzyme for G6PDH. In some embodiments, themethod further comprises detecting a change in the enzymatic activity ofthe enzyme-analyte conjugate, wherein the change in enzymatic activityis related to the amount of the analyte of interest present in thesample.

In some embodiments, the antibody is present at a concentration fromabout 0.4 μg/mL to about 1.0 μg/mL. In some embodiments, the conjugatecomprising an enzyme and the analyte of interest is present at aconcentration from about 0.4 μg/mL to about 0.5 μg/mL.

In some embodiments, the determining the presence or absence of theanalyte of interest is determined at a temperature of about 4° C. toabout 45° C. In some embodiments, the determining the presence orabsence of the analyte of interest is determined at a temperature ofabout 30° C. to about 40° C. In some embodiments, the determining thepresence or absence of the analyte of interest is determined at atemperature of about 37° C.

In some embodiments, the aqueous medium has a pH range of about 4.0 toabout 11.0. In some embodiments, the aqueous medium has a pH of about7.0. In some embodiments, the aqueous medium contains less than about20% of a polar solvent. In some embodiments, the aqueous medium containsless than about 20% of N,N-Dimethylformamide (DMF), acetonitrile,dimethylsulfoxide (DMSO), methanol, or a combination thereof.

In some embodiments, the analyte of interest is selected from the groupconsisting of a drug of abuse, a toxic chemical, an environmentalchemical, a petroleum product, a natural product, an organic compound, anutrient, a prescription and over-the-counter medication, or ametabolite, derivative, or breakdown product of any of the foregoing. Insome embodiments, the analyte of interest is a drug of abuse ormetabolite thereof. In some embodiments, the drug of abuse or metabolitethereof is selected from the group consisting of cocaine,benzoylecgonine, cocaethylene, norocaine, PCP, amphetamine,methamphetamines, cannabinoids, THC, carboxy-THC, nicotine, cotinine,benzodiazepines, heroin, codeine, morphine, 6-monoacetylmorphine (MAM),oxycodone, 3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA). In some embodiments, theanalyte of interest is selected from the group consisting of opioids,amphetamines, NSAIDS, steroids, cannabinoids, benzodiazepines,barbiturates, tricyclics, nicotine, and ephedrines, or metabolites,derivatives, or breakdown products of any of the foregoing.

Also provided herein is a method for determining the presence or absenceof an analyte of interest in a keratinized structure sample comprising:combining in an aqueous medium: an enzyme-analyte conjugate comprisingglucose-6-phosphate dehydrogenase (G6PDH) covalently linked to theanalyte of interest; an antibody specific for the analyte of interest; akeratinized structure sample; an enzyme substrate for G6PDH; and acoenzyme for G6PDH; and determining if the analyte of interest ispresent or absent in the keratinized structure sample.

Also provided herein is a method for determining the presence or absenceof an analyte of interest in a keratinized structure sample comprising:combining in an aqueous medium: an enzyme-analyte conjugate comprisingglucose-6-phosphate dehydrogenase (G6PDH) covalently linked to theanalyte of interest; an antibody specific for the analyte of interest; akeratinized structure sample; an enzyme substrate for G6PDH; and acoenzyme for G6PDH; and determining if the analyte of interest ispresent or absent in the keratinized structure sample, wherein theanalyte of interest is a drug of abuse or metabolite thereof.

In some embodiments, the keratinized structure sample is a hair sample.In some embodiments, the keratinized structure sample is a nail sample.

Also provided herein is a kit comprising: a conjugate comprising anenzyme and an analyte of interest; and an antibody specific for theanalyte of interest.

In some embodiments, the enzyme is selected from the group consisting ofglucose-6-phosphate dehydrogenase (G6PDH), alcohol dehydrogenase,glutamic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase,α-glycerol phosphate dehydrogenase, lactic dehydrogenase,glyceraldehydes-3-phosphate dehydrogenase, gluthathione reductase,quinine reductase, nitrate reductase, and glutamic dehydrogenase. Insome embodiments, the enzyme is glucose-6-phosphate dehydrogenase(G6PDH). In some embodiments, the enzyme is glucose-6-phosphatedehydrogenase (G6PDH).

In some embodiments, the antibody specific for the analyte of interestis selected from the group consisting of cocaine, benzoylecgonine,cocaethylene, norcocaine, PCP, amphetamine, methamphetamine,cannabinoids, THC, carboxy-THC, heroin, benzodiazepines, methadone,codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA).

In some embodiments, the G6PDH is obtained from a natural source. Insome embodiments, the G6PDH is a recombinant enzyme.

In some embodiments, the kit further comprises: an enzyme substrate forG6PDH; and a coenzyme for G6PDH.

In some embodiments, the analyte of interest is selected from the groupconsisting of a drug of abuse, a toxic chemical, an environmentalchemical, a petroleum product, a natural product, an organic compound, anutrient, a prescription, and over-the-counter medication, or ametabolite, derivative, or breakdown product of any of the foregoing. Insome embodiments, the analyte of interest is a drug of abuse ormetabolite thereof. In some embodiments, the drug of abuse or metabolitethereof is selected from the group consisting of cocaine,benzoylecgonine, cocaethylene, norocaine, PCP, amphetamine,methamphetamines, cannabinoids, THC, carboxy-THC, nicotine, cotinine,benzodiazepines, heroin, codeine, morphine, 6-monoacetylmorphine (MAM),oxycodone, 3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA).

In some embodiments, the analyte of interest is selected from the groupconsisting of opioids, amphetamines, NSAIDS, steroids, cannabinoids,benzodiazepines, barbiturates, tricyclics, nicotine and ephedrines, ormetabolites, derivatives, or breakdown products of any of the foregoing.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

Other features and advantages of the methods as provided herein will beapparent from the following detailed description and figures, and fromthe claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an exemplary graphical representation of a calibration curvefor cocaine obtained by plotting the results obtained in Example 6. Theconcentration of cocaine is shown on the X-axis and the absorbance at340 nm (ΔmAU) is shown on the Y-axis.

FIG. 2 is an exemplary graphical representation of a calibration curvefor opioid obtained by plotting the results obtained in Example 7. Theconcentration of opioid is shown on the X-axis and the absorbance at 340nm (ΔmAU) is shown on the Y-axis.

FIG. 3 is an exemplary graphical representation of a calibration curvefor oxycodone obtained by plotting the results obtained in Example 8.The concentration of oxycodone is shown on the X-axis and the absorbanceat 340 nm (ΔmAU) is shown on the Y-axis.

FIG. 4 is an exemplary graphical representation of a calibration curvefor PCP obtained by plotting the results obtained in Example 9. Theconcentration of PCP is shown on the X-axis and the absorbance at 340 nm(ΔmAU) is shown on the Y-axis.

FIG. 5 is an exemplary graphical representation of a calibration curvefor methamphetamine obtained by plotting the results obtained in Example10. The concentration of methamphetamine is shown on the X-axis and theabsorbance at 340 nm (ΔmAU) is shown on the Y-axis.

DETAILED DESCRIPTION

The present disclosure relates to methods and kits for a homogeneousenzyme immunoassay for the qualitative determination of analytes (e.g.,low concentration analytes) in keratinized structure (e.g., hair)samples.

Current methods used to screen for particular analytes are regulated bythe Substance Abuse mental Health Services Administration (SAMHSA).These regulations include cutoff calibrator concentrations for approvedscreening assays and these cutoffs vary depending on the sample used forscreening as well as the analyte being detected. As shown in Table A,the calibrator concentrations for many typical analytes detected in hairare 1.5- to 10-fold lower than those for oral fluid.

TABLE A Oral Fluid Calibrator Hair Calibrator Test Analyte (ng/mL)¹ TestAnalyte (ng/10 mg)² Cocaine 15 Cocaine 5 Opioids 30 Opioids 26-Acetylmorphine 3 6-Acetylmorphine 2 Phencyclidine 3 Phencyclidine 3Amphetamines 25 Amphetamines³ 3 ¹SAMHSA Proposed Guidelines Vol. 80, No.94, p. 28054-28101, 2015 ²SAMHSA Proposed Guidelines Vol. 69, No. 71, p.19673-19732 ³510(k) k051161

This significant decrease in calibrator concentrations for hair samplesas compared to oral fluid samples represents a challenge for developinga homogeneous enzyme immunoassay for hair. In addition, the hair matrixpresents potential confounding substances (e.g., cosmetic treatments,hair styling products, hair care products, deodorants) that can beextracted during extraction of the analyte of interest from the hair.These same confounding substances are not found in oral matrices. Forexample, keratinized structures such as hair maintain potentiallyconfounding substances throughout the length of the segment tested, thisis not, however, the case for liquid matrices (e.g., urine and oralfluid) where the interfering substances only include those presentduring the short time period before and during sample collection.Finally, the hair matrix is a solid matrix with very minimal analyteconcentrations present and the amount of solid hair matrix employed islimited by the amount of extraction solution. Meaning, more hair cannotalways be added to the extraction solution, thus limiting theconcentration of the drug that can be eluted.

Overall, these limitations make the final sample to use in thehomogeneous immunoassay not only extremely low in abundance but also lowin purity, posing significant obstacles to overcome while developing ahomogeneous immunoassay for hair exhibiting the required sensitivity andspecificity.

Provided herein is a method for determining the presence or absence ofan analyte of interest in a keratinized structure (e.g., hair) sample,comprising first combining in an aqueous medium: a conjugate comprisingan enzyme and the analyte of interest, an antibody specific for theanalyte of interest, and the keratinized structure (e.g., hair) sample,and then determining whether the analyte of interest is present orabsent in the keratinized structure (e.g., hair) sample.

Enzymes, Coenzymes, and Substrates

The methods provided herein use, as one component, a conjugatecomprising an enzyme and the analyte of interest.

In some embodiments, the enzyme utilizes nicotinamide adeninedinucleotide (NAD/NAD+) or nicotinamide adenine dinucleotide phosphate(NADP/NADP+) as a co-enzyme to generate NADH and NADPH, respectively.For example, the enzyme can be glucose-6-phosphate dehydrogenase(G6PDH). In some embodiments, the enzyme is an enzyme other G6PDH.Non-limiting examples of additional enzymes that are useful for thepresent disclosure and which use NAD (NAD+) as a co-enzyme and generateNADH include, alcohol dehydrogenase, glutamic dehydrogenase, malicdehydrogenase, isocitric dehydrogenase, α-glycerol phosphatedehydrogenase, lactic dehydrogenase, and glyceraldehydes-3-phosphatedehydrogenase. Non-limiting examples of additional enzymes that areuseful for the methods described herein and which use NADP (NADP+) as aco-enzyme and generate NADPH include gluthathione reductase, quininereductase, nitrate reductase, and glutamic dehydrogenase. Furthernon-limiting examples of enzymes and co-enzymes useful in the methodsprovided herein are disclosed in U.S. Pat. Nos. 4,275,149 and 4,318,980,which are each incorporated herein by reference in their entirety.

In some embodiments, the G6PDH is capable of using both NADP+ and NAD+.For example, those isolated from Leuconostoc mesenteroides, A.suboxydans, P. aeruginosa, Pseudomonas W6, H. eutrophaH-16,Hydrogenomonas facilis, Arthrobacter 7C, A. beijerickii, T.ferrooxidans, B. licheniformis, P. denitrificans, C. crescentus, L.lactis, and R. spheroides. In some embodiments, G6PDH is capable ofusing NAD+ as a preferred cofactor. For example, those isolated from P.fluorescens and one of the G6PDHs from P. multivorans. In someembodiments, the G6PDH is NAD+ specific. For example, one of the G6PDHsfrom A. xylinum. As another example, Leuconostoc mesenteroidesglucose-6-phosphate dehydrogenases are dimeric enzymes that have theability to catalyze the oxidation of D-glucose-6-phosphate toD-glucono-δ-lactone-6-phosphate by utilizing either NAD+ or NADP+. Thisproperty of using NAD+ differentiates these enzymes from human G6PDH,which utilizes only NADP+ effectively, and allows L.mesenteroides-specific G6PDH activity to be measured in the presence ofhuman G6PDH, as, for example, in human samples.

In some embodiments, the bacteria from which to select DNA encodingG6PDH are Leuconostoc and Zymomonas. For example, L. mesenteroides, L.citreum, L. lactis, L. dextranicum, and Z. mobilis. In some embodiments,the bacteria are selected from, L. mesenteroides, L. citreum, and L.lactis. In some embodiments, G6PDH from Leuconostoc is used for mutationstrategies wherein one or more cysteine residues are introduced as itdoes not contain cysteine residues.

Table 1 of U.S. Pat. No. 6,033,890, the disclosure of which isincorporated by reference in its entirety, describes exemplary strainsof various Leuconostoc species. In some embodiments, strains from whichto select G6PDH are Leuconostoc mesenteroides strain ATCC 12291,Leuconostoc citreum strain NCIMB 3351, Leuconostoc lactis strain NCDO546, and Leuconostoc dextranicum strain ATCC 19255. In some embodiments,other G6PDH suitable for use in the present disclosure include, but arenot limited to, those described in Bacillus megaterium M1286 (Heilman etal., Eur. J. Biochem. (1988) vol. 174, 485-490); Saccharomycescerevisiae (Jeffrey et al., Biochemistry, (1985) vol. 24, 666-671);Pichia jadinii (Jeffrey et al., Biochem. Biopys. Res. Comm., (1989) vol.160:3, 1290-1295), E. coli K-12 (Rowley et al., J. Bacterial., (1991)vol. 173:3, 968-977), and from human (Bhadbade et al., FEES Lett. (1987)vol. 211, 243-246).

In some embodiments, an enzyme from natural or recombinant sources orsite-directed mutants, and any isoform, site-directed mutant or amixture of isoforms and site-directed mutants may be used. For example,G6PDH from natural or recombinant sources or site-directed mutants, andany isoform, site-directed mutant or a mixture of isoforms andsite-directed mutants may be used. Several G6PDH enzymes from variousspecies are known as described herein, in U.S. Pat. No. 6,033,890 and byLevy (Adv. Enzym. (1979) vol. 48, 97-192). In some embodiments, anenzyme (e.g., G6PDH) from natural sources may be purified followingprocedures known to the skilled artisan.

In some embodiments, the enzyme is a recombinant enzyme. In someembodiments, the G6PDH is a recombinant G6PDH. The basic molecularbiological techniques employed in generating a recombinant G6PDH caninclude methods such as DNA and plasmid isolation, restriction enzymedigestion, DNA ligation, purification and characterization of DNAs bypolyacrylamide and agarose gel electrophoresis, labeling andhybridization of DNAs, Southern blotting, transformation, maintenanceand growth of bacterial strains, protein expression and proteinpurification, and other general techniques are all well known in theart. For example, general techniques of molecular biology are describedin “Molecular Cloning A Laboratory Manual” by Sambrook, J., Fritsch, E.F., and Maniatis, T. published by Cold Spring Harbor Laboratory Press,2nd edition, 1989, or “A Practical Guide to Molecular Cloning” byBernard Perbal published by John Wiley & Sons, New York, 1984. In someembodiments, the DNA encoding a G6PDH of interest is cloned into anexpression vector and transformed into a suitable host cell, whichexpresses the recombinant G6PDH. In some embodiments, the recombinantG6PDH may then be purified using methods known to the skilled artisan.Recombinant G6PDH enzymes have been described and include, but are notlimited to, G6PDHs from L. mesenteroides (Adams et al., J. Biol. Chem.,(1983) vol. 258:9, 5867-5868; Murphy et al., J. Bacterial., (1987) vol.169:1, 334-339; Lee et al., J. Biol. Chem., (1991) vol. 266:20,13028-13034); Z. mobiles (Barnell et al., J. Bacterial., (1990) vol.172:12, 7227-7240); Bacillus megaterium M1286 (Heilmann et al., Eur. J.Bio-chem., (1988) vol. 174, 485-490); and E. coli K-12 (Rowley et al. J.Bacterial., (1991) vol. 173:3, 968-977).

In some embodiments, the enzyme is a mutated enzyme. In someembodiments, the G6PDH is a mutated G6PDH. In some embodiments, G6PDHsdiffering from any naturally occurring G6PDH may be generated usingmolecular DNA cloning technologies as known in the art. In someembodiments, G6PDHs with amino acid substitutions, deletions, orinsertions, or any combination thereof may be generated (see U.S. Pat.No. 6,033,890, which is incorporated by reference in its entirety) andused in the methods of this disclosure.

In some embodiments, the enzyme is obtained from a commercial source. Insome embodiments, the Glucose-6-Phosphate Dehydrogenase (G6PDH) isobtained from a commercial source (e.g., Sigma, Biochemica, BoehringerMannheim, USE Biochemical, and OYC International Inc., which isincorporated by reference in its entirety), and its activity isdetermined either experimentally or is provided by the manufacturer. Insome embodiments, the starting activity of the G6PHD is greater thanabout 400 U/mg. For example, the starting activity of the G6PDH isgreater than about 500 U/mg, greater than about 600 U/mg, greater thanabout 700 U/mg, greater than about 800 U/mg, greater than about 900U/mg, greater than about 1,000 U/mg, greater than about 1,100 U/mg, orgreater than about 1,200 U/mg. In some embodiments the starting activityof the G6PDH is greater than about 850 U/mg.

In some embodiments, the aqueous medium used in the methods providedherein further comprises an enzyme substrate for G6PDH and a coenzymefor G6PDH. In some embodiments, the method for determining the presenceor absence of an analyte of interest in a keratinized structure (e.g.,hair) sample further comprises detecting a change in the enzymaticactivity of the enzyme-analyte conjugate, wherein the change inenzymatic activity is related to the amount of the analyte of interestpresent in the sample. In some embodiments, the enzymatic activities ofG6PDH, the G6PDH-analyte conjugate, the G6PDH-analyte conjugate withbound antibody and the G6PDH-analyte conjugate with bound antibodycompeting for analyte binding in a test sample are determined. In someembodiments, determination of enzymatic activity is dependent on asubstrate and co-enzyme for G6PDH. In some embodiments, a suitablesubstrate for G6PDH is glucose-6-phosphate (G6P). In some embodiments,suitable co-enzymes or cofactors for G6PDH are NAD (NAD+) and NADP(NADP+). In some embodiments, G6PDH converts G6P and co-enzymes into6-P-glucuronate and NADH and NADPH, respectively. In some embodiments,in order to measure G6PDH activity, G6P and NAD+ or NADP+ are added tothe aqueous medium. In some embodiments, cofactor analogs, such asthio-NAD+, thio-NADH, thio-NADP+, or thio-NADPH may also be used.

In some embodiments, substrate and co-enzyme or co-factors for G6PDH arenot labeled and the signal generated by G6PDH, i.e., the amount of NADPHor NADH, is measured in a spectrophotometer as described herein. In someembodiments, the substrate and or co-enzymes may be labeled and thesignal generated by G6PDH may be detected by other means, depending onthe label, such as by measuring change in fluorescence or scintillationcount, or the like.

Provided herein, is a method for determining the presence or absence ofan analyte of interest in a keratinized structure (e.g., hair) samplecomprising, combining in an aqueous medium an enzyme-analyte conjugatecomprising glucose-6-phosphate dehydrogenase (G6PDH) covalently linkedto the analyte of interest, an antibody specific for the analyte ofinterest, a keratinized structure (e.g., hair) sample, an enzymesubstrate for G6PDH, and a coenzyme for G6PDH, and determining if theanalyte of interest is present of absent in the keratinized structure(e.g., hair) sample using any suitable method including those describedherein. In some embodiments, the keratinized structure sample is a hairsample. In some embodiments, the keratinized structure sample is a nailsample. For example, the nail sample can be a toenail sample or afingernail sample.

Analytes

In some embodiments, the disclosure provides a conjugate comprising anenzyme and the analyte of interest. In some embodiments, an analyte ofthe disclosure can be any substance, compound or composition whosepresence or concentration in a sample or specimen is to be determined.

In some embodiments, analytes can be polyepitopic or monoepitopic. Insome embodiments, monoepitopic analytes will have a molecular weightfrom about 100 to 5,000 molecular weight, from about 500 to 3,000molecular weight, or from about 125 to 2,000. In some embodiments,polyepitomic analytes will have a molecular weight of at least 5,000molecular weight, at least about 7,500 molecular weight, or at leastabout 10,000 molecular weight. In some embodiments, poly amino acidanalytes of interest may include proteins, polypeptides and peptides andcan be from about 5,000 to 5,000,000 molecular weight, from about 10,000to 2,500,000 molecular weight, or from about 20,000 to 1,000,000molecular weight.

In some embodiments, the analyte of interest is selected from the groupconsisting of a drug of abuse, a toxic chemical, an environmentalchemical, a petroleum product, a natural product, an organic compound, anutrient, a prescription and over-the-counter medication, or ametabolite, derivative, or breakdown product of any of the foregoing. Insome embodiments, the analyte of interest is a drug of abuse ormetabolite thereof. In some embodiments, the the drug of abuse ormetabolite thereof is selected from the group consisting of cocaine,benzoylecgonine, cocaethylene, norocaine, phencyclidine (PCP),amphetamine, methamphetamines, cannabinoids, THC, carboxy-THC, heroin,codeine, morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA). In some embodiments, theanalyte of interest is selected from the group consisting of opioids,amphetamines, NSAIDS, steroids, cannabinoids, benzodiazepines,barbiturates, tricyclics, and ephedrines, or metabolites, derivatives,or breakdown products of any of the foregoing.

In some embodiments, the analyte of interest is selected from the groupconsisting of cocaine, morphine, 6-Acetyl morphine (6-AM), hydrocodone,oxycodone, oxymorphone, methamphetamine,3,4-methylenedioxymethamphetamine (MDMA), and phencyclidine (PCP).

Non-limiting examples of analytes of interest that may be used with themethods disclosed herein are licit and illicit drugs, sugars (including,but not limited to, mono-, di-, and poly-carbohydrates), amino acids,peptides, nucleic acids, nucleosides, nucleotides, vitamins, hormones,steroids, antibiotics, bacterial or microbial antigens, toxins, chemicaland biological warfare agents, pesticides, herbicides, industrialchemicals and pollutants and include analogs, derivatives andmetabolites of such compounds. Additional non-limiting examples ofanalytes of interest that may be used with the methods disclosed hereinare opium, the opioid analgesics, alkaloids, catecholamines,epinephrine, amphetamines, barbiturates, benzodiazepines, cardiac drugs,anti-seizure drugs, immunosuppressants, tetrahydrocannabinol (THC, theactive ingredient in marijuana), cocaine, cocaine metabolite(benzoylecgonine), crack, inhalants (e.g., amyl or butyl nitrates),phencyclidine (PCP), 3,4-methylendioxymethamphetamine (MDMA, or ecstasy)and its related compounds such as 3,4-methylendioxyamphetamine (MDA) and3,4-methylenedioxyethylamphetamine (MDEA), ketamine, lysergic aciddiethylamide (LSD), γ-hydroxybutyrate (GHB), methaqualone (also calledquinazolinone), tranquilizers, alcohol, etc. In some embodiments,included in these classes are analogs, metabolites, and derivatives ofsuch compounds. In some embodiments, the analyte of interest is anopioid analgesic. Non-limiting examples or opioid analgesics includeopium, morphine, heroin, codeine, dihydrocodeine (DF-118),hydromorphone, fentanyl, oxycodone, buprenorphine, butorphanol,nalbuphine, methadone, physeptone, pethidine, dioconal, palium,dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon®),dextroproxyphene, pethidine, methylphenidate (Ritalin), acetylmethadol,and include analogs, metabolites, and derivatives of such opioidanalgesics.

In some embodiments, the analyte of interest is an alkaloid.Non-limiting examples of alkaloids that may be used with the methodsdisclosed herein are steroid alkaloids, the iminazolyl alkaloids, theisoquinoline alkaloids, the quinoline alkaloids (including quinine), thediterpene alkaloids, and include analogs, metabolites, and derivativesof such alkaloids.

In some embodiments, the analyte of interest is a catecholamine.Non-limiting examples of catecholamines that may be used with themethods disclosed herein are cotamine, narceine, noscapine andpapaverine epinephrine, L-dopa, ephedrine, and include analogs,metabolites, and derivatives of such catecholamines.

In some embodiments, the analyte of interest is an amphetamine or arelated compound. Non-limiting examples of amphetamines and relatedcompounds include amphetamine, methamphetamine, and the like. In someembodiments, included are analogs, metabolites, and derivatives of suchamphetamines or related compounds.

In some embodiments, the analyte of interest is a barbiturate.Non-limiting examples of barbiturates include veronal, pentobarbital(Nembutal), amobarbital, secobarbital (Seconal), phenobarbital,thiopental, and include analogs, metabolites, and derivatives of suchbarbiturates. In some embodiments, the analyte of interest is abenzodiazepine. Non-limiting examples of benzodiazepines includeDiazepam (Valium), chlordiazepoxide (Librium), Nitrazepam (Mogodon),Temazepam, and include analogs, metabolites, and derivatives of suchbenzodiazepines. In some embodiments, analyte is a hallucinogen.Non-limiting examples of hallucinogens include mescaline, psilocybin,psilocin, dextromoramide (Palfium), LSD, MDA(3,4-methylenedioxyamphetamine), Ecstacy (MDMA,3,4-methylenedioxymethamptamine), MDEA(3,4-methylenedioxyethylamphetamine), PMA (para-methoxyamphetamine),PMMA (para-methoxymethylamphetamine), PCP (phencyclidine), and includeanalogs, metabolites, and derivatives of such hallucinogens.

In some embodiments, the analyte of interest is a cardiac drug.Non-limiting examples of cardiac drugs include digoxin, digitoxin,N-acetyl procainamide, procainamide, quinidine, lidocaine, and includeanalogs, metabolites, and derivatives of such cardiac drugs. In someembodiments, the analyte is an anti-seizure drug. Non-limiting examplesof anti-seizure drugs include phenytoin, Phenobarbital, primidone,valproic acid, ethosuximide, carbamazepine, and include analogs,metabolites, and derivatives of such anti-seizure drugs. In someembodiments, the analyte is an immunosuppressant. Non-limiting examplesof immunosuppressant include MPA (mycophenolic acid), cyclosporine,rapamycin (sirolimus), FK506 (tacrolimus), and include analogs,metabolites, and derivatives of such immunosuppressants.

Further non-limiting examples of an analyte of interest contemplated bythe methods as provided herein include vitamins and diet supplementssuch as folic acid, thiamine, Vitamin B12, biotin, Vitamin A, Vitamin B,Vitamin C, Vitamin D, Vitamin E, Vitamin K, tranquilizers such asmeprobamate, and tricyclic anti-depressants, food supplements and otherperformance-enhancing agents, and include analogs, metabolites, andderivatives of such compounds.

In some embodiments, the analyte of interest is an amino acid.Non-limiting examples of amino acids include glycine, alanine, serine,histidine, methionine, and include analogs, metabolites, and derivativesof such amino acids. In some embodiments, the analyte of interest is anantibiotic. Non-limiting examples of antibiotics include penicillin,chloromycetin, actinomycin, tetracycline, terramycin, gentamycin,kanamycin, tobromycin, tobramycin, netilmicin, amikacin, vancomycin, andinclude analogs, metabolites, and derivatives of such antibiotics. Insome embodiments, the analyte of interest is a microbial antigen.Non-limiting examples of microbial antigens include Clostridiumdifficile antigen, Toxin A, aflatoxin B 1, and include analogs,metabolites, and derivatives of such microbial antigens.

In some embodiments, the analyte of interest is a hormone. Non-limitingexamples of hormones include thyroid hormones (T3 and T4), thyroxine,thyroid stimulating hormone, estrogen, progesterone, testosterone,prolactin, follicle stimulating hormone, chorionic gonadotropin,luteinizing hormone, include analogs, metabolites, and derivatives ofsuch hormones. In some embodiments, the analyte is a steroid.Non-limiting examples of steroids include various estrogens andandrogens such as ethynylestradiol, testosterone, androsterone, andinclude analogs, metabolites, and derivatives of such steroids.

In some embodiments, the analyte of interest is a chemical or biologicalwarfare agent. Non-limiting examples of chemical or biological warfareagents include mustard gas, Sarin, Tabun, Bacillus anthracis (Anthrax)antigens, Smallpox viral antigens, and include analogs, metabolites, andderivatives of such chemical or biological warfare agents. In someembodiments, the analyte of interest is an industrial chemical.Non-limiting examples of industrial chemicals include flavoring agents,food additives, preservatives, food contaminants, air and chemicalpollutants, pesticides, herbicides, and include analogs, metabolites,and derivatives of such industrial chemicals.

Provided herein is a method for determining the presence or absence ofan analyte of interest in a keratinized structure (e.g., hair) samplecomprising, combining in an aqueous medium an enzyme-analyte conjugatecomprising glucose-6-phosphate dehydrogenase (G6PDH) covalently linkedto the analyte of interest, an antibody specific to the analyte ofinterest, a keratinized structure (e.g., hair) sample, an enzymesubstrate for G6PDH, and a coenzyme for G6PDH, and determining if theanalyte of interest is present or absent in the keratinized structure(e.g., hair) sample, wherein the analyte of interest is a drug of abuseor metabolite thereof.

Conjugation

In some embodiments, the enzyme (e.g., G6PDH) is covalently linked tothe analyte of interest. In some embodiments, the enzyme (e.g., G6PDH)is covalently linked to an analyte that is identical or substantiallyidentical to the specific analyte of abuse to be measured. In someembodiments, the substantially identical analyte has greater than about80% cross-reactivity to the analyte. For example, the substantiallyidentical analyte has greater than about 85%, greater than about 90%,greater than about 95%, or greater than about 98% cross-reactivity tothe analyte.

In some embodiments, conjugation can be achieved via conventionalchemical reactions as known in the art. For example, a reaction tocoupling an analyte (or a hapten) to the enzyme (e.g., G6PDH) is throughthe formation of a peptide bond (—CONH₂). Non-limiting examples includeusing a carboxyl (—COOH) group on an analyte (or a hapten) to react withan amino group (—NH₂) on the G6PDH enzyme (Biochem. and Biophys. Res.Comm., (1989) vol. 160:3, 1290-1295). Glucose-6-phosphate dehydrogenasefrom Leuconostoc mesenteroides is reported to contain a total of 38lysine residues (Levy, Adv. Enzym, (1979) vol. 48, 97-192; FEBS Lett.211:2, 243-246, 1987). In some embodiments, under appropriate couplingconditions, the €-amino groups from these lysine moieties can bemodified readily. In some embodiments, multiple molecules of an analyte(or hapten) and/or a plurality of analytes (or haptens) can beconjugated to each molecule of the enzyme (e.g., G6PDH).

In some embodiments, analytes are capable of binding directly to theenzyme (e.g., G6PDH). In some embodiments, analytes are not capable ofcovalent binding directly. In some embodiments, analytes not capable ofcovalent binding directly are rendered capable of covalently binding tothe enzyme (e.g., G6PDH) by the addition of a linking group that cancovalently bind to a group on the enzyme (e.g., G6PDH) (e.g., to anamino, hydroxyl, carboxyl or mercapto group). Non-limiting examples oflinking groups include amino acids having one or more free amino or freehydroxyl groups, carbonyl, thiocarbonyl, or carboxyl groups, orcompounds containing such groups. Additional non-limiting examples oflinking groups include N-hydroxysuccinimide and other succinimide ormaleimide-containing moieties, and1-(3-dimethylpropyl)-3-ethylcarbodiimide. Further non-limiting examplesof linking groups are found in U.S. Pat. No. 3,817,837, which isincorporated by reference in its entirety.

In some embodiments, linking groups suitable for use in this disclosureinclude compounds of about less than 50 atoms other than hydrogens,about less than 20 atoms other than hydrogens, about less than 6 atomsother than hydrogens and having a chain (i.e., a spacer) of not morethan about 35, less than about 15, less than about 10, or less thanabout 5 atoms in length. Non-limiting examples of linking groups usablein preparing conjugates for this disclosure include bifunctionalcrosslinking or coupling agents (i.e., molecules containing two reactivegroups or “ends”), which may be tethered by a spacer of variable length.In some embodiments, the reactive ends can be any of a variety offunctionalities including, but not limited to, amino reacting ends suchas N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes,epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroarylhalides; and thiol reacting ends such as pyridyl disulfides, maleimides,and thiophthalimides. In some embodiments, the heterobifunctionalcrosslinking reagents have two different reactive ends (e.g., anamino-reactive end and a thiol-reactive end) while homobifunctionalreagents that are usable in preparing the conjugates of this disclosurehave two similar reactive ends. Non-limiting examples of such includebismaleimidohexane (BMH), which permits the cross-linking ofsulfhydryl-containing compounds, and NHS homobifunctional crosslinkerssuch as disuccinimidyl suberate (DSS) as well as the water solubleanalogs, sulfo-NHS esters. Additional non-limiting examples of suitablelinking groups for use in the present disclosure include maleimido-NHSactive esters coupling agents such asm-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS); succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); succinimidyl4-(p-maleimidophenyl)butyrate (SMPB) and derivatives thereof, includingsulfosuccinimidyl derivatives such as sulfosuccinimidyl4-(N-maleimido-methyl) cyclohexane-1-carboxylate (sulfo-SMCC);m-maleimidobenzoyl-sulfosuccinimide ester (sulfo-MBS) andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) (Pierce).Non-limiting examples of suitable heterobifunctional reagents includecommercially available active halogen-NETS active esters coupling agentssuch as N-succinimidyl bromoacetate and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) and the sulfo-succinimidylderivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate(sulfo-SIAB) (Pierce). Another non-limiting example of coupling agentsis the heterobifunctional and thiol cleavable agents such asN-succinimidyl 3-(2-pyridyidithio-)propionate (SPDP) (Pierce).

Non-limiting examples of commercially available homobifunctionalcross-linking reagents include the imidoesters such as dimethyladipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride(DMP); and dimethyl suberimidate dihydrochloride (DMS).

In some embodiments, the conjugates are prepared by contacting theactivated analyte or hapten with a buffered solution of enzyme (e.g.,G6PDH) under conditions for formation of such conjugates. In someembodiments, conditions for forming such conjugates include atemperature of from about 2° C. to about 25° C., a pH of from about 5 toabout 10, and a contact time of from less than an hour to several days.

In some embodiments, the enzyme-analyte conjugate is purified afterconjugation. For example, suitable purification procedures are known inthe art and non-limiting examples include dialysis againstaqueous/organic and aqueous solutions such as water/DMF or water, or bygel filtration chromatography on supports such as Sephadex, and thelike.

Deactivation of G6PDH

In some embodiments, covalently linking an analyte of interest to theenzyme (e.g., G6PDH) leads to a change of enzymatic activity (e.g.,G6PDH enzymatic activity), which can be measured using the methods ofthis disclosure. In some embodiments, this change of enzymatic activityis a decrease of enzymatic activity by the enzyme-analyte conjugate(e.g., G6PDH-analyte conjugate) when compared to the activity of thenative enzyme (e.g., G6PDH), i.e., the enzyme (e.g., G6PDH), which isnot conjugated to an analyte. In some embodiments, the decrease ofenzymatic activity due to the covalent linking of an analyte to theenzyme (e.g., G6PDH) is referred to as deactivation. In someembodiments, covalent linkage of the analyte to the enzyme (e.g., G6PDH)results in permanent deactivation of the enzyme.

In some embodiments, the ratio of analyte conjugated to the enzyme(e.g., G6PDH) is dependent on the desirable % of deactivation of theenzyme (e.g., G6PDH) and the desirable % inhibition of the resultingenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) exhibited uponbinding to the specific antibody. In some embodiments, the extent ofdeactivation will be proportional to the extent of conjugation. In someembodiments, the extent of deactivation may be controlled, for example,by measuring enzymatic activity on samples taken at various times ofconjugation.

In some embodiments, the inhibition of the enzyme-analyte conjugate(e.g., G6PDH-analyte conjugate) is from about 20% to about 50%. In someembodiments, the enzyme (e.g., G6PDH) is deactivated by from about 20%to about 60% and the enzyme activity of the deactivated enzyme-analyteconjugate (e.g., G6PDH-analyte conjugate) is further inhibited by fromabout 40% to about 80%. In some embodiments, the enzyme (e.g., G6PDH) isdeactivated by from about 30% to about 65% and the enzyme activity ofthe deactivated enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate)is further inhibited by from about 40% to about 85%.

In some embodiments, the higher the specific activity of theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate), the higher theassay sensitivity. In some embodiments, the enzyme-analyte conjugate(e.g., G6PDH-analyte conjugate) has a minimum specific activity. In someembodiments, with about 10% deactivation, the specific activity of theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is in the rangeof about 450 U/mg to about 1,800 U/mg, in the range of about 540 U/mg toabout 1,350 U/mg, or in the range of about 630 units/mg to about 900units/mg. In some embodiments, enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 720U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 810U/mg.

In some embodiments, with about 20% deactivation, the specific activityof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is inthe range of about 400 U/mg to about 1,600 U/mg, in the range of about480 U/mg to about 1,200 U/mg, or in the range of about 560 U/mg to about800 U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 640U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 720U/mg.

In some embodiments, with about 30% deactivation, the specific activityof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is inthe range of about 350 U/mg to about 1,400 U/mg, in the range of about420 U/mg to about 1,050 U/mg, or in the range of about 490 U/mg to about700 U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 560U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 630U/mg.

In some embodiments, with about 40% deactivation, the specific activityof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is inthe range of about 30 U/mg to about 1,200 U/mg, in the range of about360 U/mg to about 900 U/mg, or in the range of about 420 U/mg to about600 U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 480U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 540U/mg.

In some embodiments, with about 50% deactivation, the specific activityof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is inthe range of about 250 U/mg to about 1,000 U/mg, in the range of about300 U/mg to about 750 U/mg, or in the range of about 350 U/mg to about500 U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 400U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 450U/mg.

In some embodiments, with about 60% deactivation, the specific activityof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is inthe range of about 200 U/mg to about 800 U/mg, in the range of about 240U/mg to about 600 U/mg, or in the range of about 280 U/mg to about 400U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 320U/mg. In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) has a specific activity of at least about 360U/mg.

Antibodies

In some embodiments, antibodies used in this disclosure include one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. Non-limiting examples ofimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. In some embodiments, immunoglobulin light chainsare classified as either kappa or lambda. In some embodiments,immunoglobulin heavy chains are classified as gamma, mu, alpha, delta,or epsilon, which define immunoglobulin classes, IgG, IgM, IgA, IgD andIgE, respectively.

In some embodiments, a typical immunoglobulin (antibody) structural unitis known to comprise a tetramer. In some embodiments, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). Insome embodiments, the N-terminus of each chain defines a variable regionof about 100 to 110 or more amino acids primarily responsible forantigen recognition. In some embodiments, the terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

In some embodiments, antibodies may exist as intact immunoglobulins oras a number of well-characterized fragments produced by digestion withvarious peptidases. For example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)₂, a dimer ofFab, which itself is a light chain joined to V_(H)-C_(H1) by a disulfidebond. In some embodiments, the F(ab)₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab)2 dimer into a Fab monomer. In some embodiments, theFab monomer is essentially a Fab with part of the hinge region (see,Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for amore detailed description of other antibody fragments). In someembodiments, various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab fragments may be synthesized de nova either chemically or byutilizing recombinant DNA methodology.

In some embodiments, the term antibody, also includes antibody fragmentseither produced by the modification of whole antibodies or synthesizedde nova using recombinant DNA methodologies. Non-limiting examples ofantibodies include single chain antibodies (antibodies that exist as asingle polypeptide chain), single chain Fv antibodies (sFv or scFv) inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide.In some embodiments, the single chain Fv antibody is a covalently linkedV_(H)-V_(L) heterodimer, which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker (Huston, et al. (1988) Proc. Natl.Acad. Sci. USA, 85: 5879-5883). In some embodiments, while the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. In some embodiments, functionalantibody molecules can be expressed on the surface of filamentous phagewere single-chain Fv's (scFv), however, alternative expressionstrategies have also been successful. For example, Fab molecules can bedisplayed on phage if one of the chains (heavy or light) is fused to g3capsid protein and the complementary chain exported to the periplasm asa soluble molecule. In some embodiments, the two chains can be encodedon the same or on different replicons; as long as the two antibodychains in each Fab molecule assemble post-translationally and the dimerincorporated into the phage particle via linkage of one of the chains tog3p (see, e.g., U.S. Pat. No. 5,733,743). In some embodiments, the scFvantibodies and a number of other structures converting the naturallyaggregated, but chemically separated light and heavy polypeptide chainsfrom an antibody V region into a molecule that folds into a threedimensional structure substantially similar to the structure of anantigen-binding site are known to those of skill in the art (see e.g.,U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778).

In some embodiments, antibodies include all those that have beendisplayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiteret al. Protein Eng., (1995) vol. 8, 1323-1331). In some embodiments,antibodies can also include diantibodies, miniantibodies, humanizedantibodies, or chimeric antibodies.

In some embodiments, the antibody specific for the analyte of interestis specific for an analyte selected from the group consisting ofcocaine, benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine,methamphetamine, cannabinoids, THC, carboxy-THC, heroin, codeine,morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA). In some embodiments, theantibody is detectably labeled. In some embodiments, the antibody isdetectably labeled with a fluorescent label, a luminescent label, aradioactive label, or an enzymatic label. In some embodiments, theluminescent label is a chemiluminescent label or a bioluminescent label.

Homogeneous Enzyme Immunoassay (HEIA)

In some embodiments, the method for determining the presence or absenceof an analyte of interest further comprises detecting a change in theenzymatic activity of the enzyme-analyte conjugate, wherein the changein enzymatic activity is related to the amount of the analyte present inthe sample. In some embodiments, the enzyme (e.g., G6PDH) can be furtherinhibited by binding of an analyte-specific antibody to theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate). In someembodiments, this inhibition is reversible; addition of a samplecontaining the analyte of interest to the antibody-enzyme conjugate(e.g., antibody-G6PDH conjugate) will result in release of the antibodyfrom the enzyme (e.g., G6PDH) conjugate, thus restoring enzyme activity.In some embodiments, measurement of the increase in enzyme (e.g., G6PDH)activity upon addition of the sample containing the analyte of interestis proportional to the concentration of the analyte in the sample. Insome embodiments, the assay is based on competition between theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) and the freeanalyte of interest in the sample for a fixed amount of antibody bindingsites.

In some embodiments, in the absence of analyte(s) in the sample, theantibody remains bound to the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate), with no change in enzyme activity. In someembodiments, when the analyte of interest is present in the sample, theantibody binds to analyte and the enzymatic activity of theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) is increased inproportion to the concentration of analyte in the sample. In someembodiments, activity of the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) is monitored by measuring the reduction of NADor NADP to NADH or NADPH, respectively, at 340 nm. In some embodiments,measuring the signal produced by reduction of NAD to NADH by theenzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) can be appliedto automated clinical analyzers. For example, such automated clinicalanalyzers include, but are not limited to, the Roche Cobas seriesanalyzers and Beckman Coulter AU series analyzers. In some embodiments,analyzers capable of maintaining constant temperature in the reactionchamber, pipetting 5 to 75 μL of sample, mixing reagents, measuringenzyme rates at 340 nm and timing the reaction accurately can be used toperform the method of the disclosure.

In some embodiments, the inhibition of the enzyme-analyte conjugate(e.g., G6PDH-analyte conjugate) used in the HEIA methods as providedherein is from about 20% to about 50%. In some embodiments, theinhibition of the enzyme-analyte conjugate (e.g., G6PDH-analyteconjugate) used in the HEIA methods as provided herein is from about 30%to about 40%. In some embodiments, the inhibition of the enzyme-analyteconjugate (e.g., G6PDH-analyte conjugate) used in the HEIA methods asprovided here is about 40%.

In some embodiments, the solvent for the homogeneous assay is an aqueousmedium containing less than about 20% of other polar solvents including,but not limited to, N,N-Dimethylformamide (DMF), acetonitrile,dimethylsulfoxide (DMSO) or methanol.

In some embodiments, the pH for the assay is between about 4.0 and about11.0. In some embodiments, the pH for the assay is between about 5.0 andabout 10.0, about 6.0 and about 9.0, or about 6.5 and about 8.5. In someembodiments, the pH for the assay is about 7.0 pH.

In some embodiments, the acceptable temperature for the assay is betweenabout 4° C. to about 45° C. In some embodiments, the acceptabletemperature for the assay is between about 30° C. to about 40° C. Insome embodiments, the acceptable temperature for the assay is about 37°C.

In some embodiments, the order of addition of reagents in the assay isnot critical. In some embodiments, the hair extract is combined with areagent solution (referred to as Antibody Buffer, see Table 2)consisting of antibody, substrate and cofactor for the enzyme (e.g.,G6PDH), followed by incubation. In some embodiments, the enzyme-analyteconjugate (e.g., G6PDH-analyte conjugate) is then added, and measurementof the enzyme-analyte conjugate (e.g., G6PDH-analyte conjugate) activityis performed.

In some embodiments, the duration of the incubation period isempirically determined. In some embodiments, the incubation period isabout 30 seconds to about 5 minutes. In some embodiments, the incubationperiod is about 2 minutes to about 4 minutes. In some embodiments, theincubation period is about 3.6 minutes.

In some embodiments, in order to accurately measure the analyteconcentration in a hair extract, the signal (expressed as ΔmA, or changein milliabsorbance) generated between a negative calibrator, typically acalibrator with 0 ng of analyte per 10 mg hair and a cutoff calibrator,such as a calibrator with 2 ng analyte/10 mg hair by enzyme-analyteconjugate (e.g., G6PDH-analyte conjugate) should be at about 40-100 ΔmA(fixed mode). In some embodiments, the enzyme-analyte conjugate (e.g.,G6PDH-analyte conjugate) generates about 40-100 ΔmA (fixed mode). Insome embodiments, equation 1 states the relationship between signalintensity, enzyme activity and reaction volume.

Enzyme Activity=ΔAU×Vt/NADH×VR2   Equation 1:

Where the AAU is the signal generated by the enzyme (e.g., G6PDH), Vt isthe total reaction volume in milliliter (mL) including the sample, R1(Antibody Buffer, Table 2) and R2 (Enzyme Buffer, Table 3) reagents,NADH is the extinction coefficient of NADH, corresponding to 6.22 andVR2 is the volume of R2 (Enzyme Buffer, Table 3) in milliliter.

In some embodiments, to meet the volume of automated analyzers, thevolume should be less than about 250 μL per assay, including sample, R1(Antibody Buffer, Table 2) and R2 (Enzyme Buffer, Table 3) volume. Insome embodiments, 25 μL of sample, 75 μL of R1 (Antibody Buffer, Table2) and 25 μL of R2 (Enzyme Buffer, Table 3) is added to generate the 40ΔmA of signal, resulting in the required enzymatic activity being 0.032U/mL. In some embodiments, the calculated enzyme activity of 0.032 U/mLis the effective enzyme amount required to generate 40 ΔmAU between theinhibited (negative/no analyte) and the reversibly inhibited(calibrator).

Kits for Determining Analyte in a Keratinized Structure (e.g., Hair)Sample Extract

Provided herein are kits, comprising a conjugate comprising an enzymeand an analyte of interest and an antibody specific for the analyte ofinterest. In some embodiments, the kits provided are used for testingthe presence and accurately determining the amount of analyte in akeratinized structure (e.g., hair) sample extract. In some embodiments,the kits of the disclosure may contain one or more of the followingcomponents: an enzyme-analyte conjugate comprising G6PDH covalentlylinked to an analyte of interest, an antibody reactive to the analyte,an enzyme substrate for G6PDH, a co-enzyme for G6PDH, a buffer,calibrators or quality controls, and an instruction manual on how toperform the HEIA. In some embodiments, the keratinized structure sampleis a hair sample. In some embodiments, the keratinized structure sampleis a nail sample. For example, the nail sample can be a toenail sampleor a fingernail sample.

In some embodiments, the antibody specific for the analyte of interestis specific for a drug of abuse or a metabolite thereof.

In some embodiments, the analyte of interest is selected from the groupconsisting of a drug of abuse, a toxic chemical, an environmentalchemical, a petroleum product, a natural product, an organic compound, anutrient, a prescription and over-the-counter medication, or ametabolite, derivative, or breakdown product of any of the foregoing. Insome embodiments, the analyte of interest is a drug of abuse ormetabolite thereof. In some embodiments, the drug of abuse or metabolitethereof is selected from the group consisting of cocaine,benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine,methamphetamines, cannabinoids, THC, carboxy-THC, heroin, codeine,morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA). In some embodiments, theanalyte of interest is selected from the group consisting of opioids,amphetamines, NSAIDS, steroids, cannabinoids, benzodiazepines,barbiturates, tricyclics, and ephedrines, or metabolites, derivatives,or breakdown products of any of the foregoing.

In some embodiments, the antibody specific for the analyte of interestis specific for an analyte selected from the group consisting ofcocaine, benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine,methamphetamine, cannabinoids, THC, carboxy-THC, heroin, codeine,morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA).

In some embodiments, reagents and compositions useful in the methods ofthe disclosure are provided in a packaged combination. In someembodiments, the reagents or compositions may be in the same or inseparate containers depending on cross-reactivity and/or stability ofthe reagents. In some embodiments, the reagents may be in lyophilizedform. In some embodiments, when reagents are provided as lyophilizedpowders, dissolving agents are included so that upon dissolution, theresulting reagent solution will have the correct concentration.

EXAMPLES

The methods as provided herein are further described in the followingexamples, which do not limit the scope of the disclosure described inthe claims.

Example 1: Calibrators and Assay Reagents

Table 1 provides the calibrators and controls for each analyte. Thecalibrators and controls are prepared by spiking the analyte intonegative hair extract buffer (e.g., 10 mM phosphate buffer, 0.0075%Tween, pH 2.4). The calibrators, controls and analyte samples are thenheated to about 80° C. for a period of about 3 hours.

TABLE 1 Calibrators and Controls Cutoff −50% Control Calibrator +100%Control Analyte (ng/10 mg) (ng/10 mg) (ng/10 mg) Cocaine 2.5 5 10Morphine 1 2 4 PCP 1.5 3 6 Methamphetamine 1.5 3 6 Oxycodone 1 2 4

Tables 2 and 3 provide the composition of Antibody Buffer and EnzymeBuffer, respectively, which were used in the provided examples.

TABLE 2 Antibody Buffer Component Amount Tris (pH 5.4) 20 mM G6P 40 mMNAD 35 mM NaCl 0.5% NaN₃ 0.09% BSA 0.1%

The antibody was formulated into Antibody Buffer at concentrations from0.4 μg/mL to 1.0 μg/mL.

TABLE 3 Enzyme Buffer Component Amount Tris (pH 8.2) 50 mM NaCl 0.9%NaN₃ 0.09% BSA 0.1%

The G6PDH-analyte conjugate was formulated into Enzyme Buffer atconcentrations from 0.4 μg/mL to 0.5 μg/mL. The G6PDH-analyte conjugatewas formulated into Enzyme Buffer to yield a cutoff calibrator ΔmAU of40 to 100 at 37° C. in a fixed mode setting on the chemistryautoanalyzer.

Example 2: Assay Protocol

Seventy-five microliters of antibody reagent (diluted in AntibodyBuffer) was incubated with 25 μL to 30 μL of calibrator or sample,followed by addition of 25 μL of G6PDH-analyte conjugate (diluted inEnzyme Buffer) at 37° C. The reaction was incubated at 37° C., and theabsorbance monitored at 340 nm. The reaction was monitored for a timeperiod of 216 seconds.

The absorbance between the first measurement and second measurement wasused to calculate the ΔmAU. The maximum rate of the enzyme wascalculated by substituting Antibody Buffer containing G6P and NAD alonefor the Antibody Buffer containing G6P, NAD and antibody.

Example 3: Preparation of G6PDH-Analyte Conjugate

Haptens (e.g., cocaine, morphine, methamphetamine, phencyclidine,oxycodone) were synthesized in-house using methods known to thoseskilled in the art. All haptens were prepared for coupling to G6PDH viaformation of an activated ester using Sulfo-N-Hydroxysuccinimide and1-(3-Dimethylpropyl)-3-Ethylcarbodiimide in anhydrous DMF.

G6PDH was purchased as a lyophilized powder and resuspended to aconcentration of 1 mg/mL using 10 mM Tris buffer, pH 7.4.

The activated ester of the hapten was added to the solution ofresuspended G6PDH at 4° C., followed by rotating overnight at the sametemperature. After this time period, the G6PDH-antigen conjugate wastransferred to a dialysis cassette and dialyzed against buffer B (Table3) that does not contain BSA. The conjugation was typically carried outat a 5-fold molar excess of antigen to the number of lysine residues permole of G6PDH.

Example 4: Preparation of G6PDH-Methamphetamine Conjugate

G6PDH with a starting activity of 870 U/mg was purchased from acommercial source. G6PDH (1 mg) was conjugated with methamphetamineantigen; after purification, 0.5 mg of G6PDH-methamphetamine wasisolated. The G6PDH-methamphetamine conjugate was reversibly inhibitedby an antibody reactive to methamphetamine. The G6PDH-methamphetamineconjugate was formulated at 0.5 μg/mL for the immunoassay. In a typicalimmunoassay, 25 μL of sample, 75 μL of methamphetamine antibody and 25μL of G6PDH-methamphetamine conjugate were used.

Example 5: Preparation of G6PDH-Oxycodone Conjugate

G6PDH with a starting activity of 891 U/mg was purchased from acommercial source. G6PDH (1 mg) was conjugated with oxycodone antigen;after purification 0.65 mg of G6PDH-oxycodone was isolated. TheG6PDH-oxycodone conjugate was reversibly inhibited by an antibodyreactive to oxycodone. The G6PDH-oxycodone conjugate was formulated at0.4 μg/mL for the immunoassay. In a typical immunoassay, 25 μL ofsample, 75 μL of oxycodone antibody and 25 μL of G6PDH-oxycodone wereused.

Example 6: Calibration and Determination of Cocaine in Hair Extract byHomogeneous Enzyme Immunoassay

The data depicted in Table 4 and FIG. 1 were obtained using the protocoldescribed in Example 1 for detecting cocaine in a hair extract sample byhomogeneous enzyme immunoassay using cocaine antibody, G6PDH-cocaineconjugate, and a hair extract calibrator and controls.

TABLE 4 Cocaine Conc. (ng/10 mg hair) ΔmAU 0 0.525 1.25 24.5 2.5 47.2 587.2 10 141.4

Example 7: Calibration and Determination of Opioids (e.g., Morphine,Codeine, 6-Acetylmorphine) in Hair Extract by Homogeneous EnzymeImmunoassay

The data depicted in Table 5 and FIG. 2 were obtained using the protocoldescribed in Example 1 for detecting opioids in a hair extract sample byhomogeneous immunoassay using opioid antibody, G6PDH-opioid conjugate,and a hair extract with morphine calibrator and morphine controls. Theassay had >80% cross reactivity with codeine and 6-acetylmorphine whenusing morphine as calibrator.

TABLE 5 Opiod Conc. (ng/10 mg hair) ΔmAU 0 2.3 1.0 34.7 2.0 57.9 4.0111.9

Example 8: Calibration and Determination of Oxycodone/Hydrocodone inHair Extract by Homogeneous Enzyme Immunoassay

The data depicted in Table 6 and FIG. 3 were obtained using the protocoldescribed in Example 1 for detecting oxycodone/hydrocodone in a hairextract sample by homogeneous immunoassay using oxycodone antibody,G6PDH-oxycodone conjugate, and a hair extract with oxycodone calibratorand oxycodone controls. The assay had >80% cross reactivity withhydrocodone when using oxycodone as calibrator.

TABLE 6 Oxycodone Conc. (ng/10 mg hair) ΔmAU 0 6.2 1 48.7 2 88.7 3 123.54 157.5

Example 9: Calibration and Determination of PCP in Hair Extract byHomogeneous Enzyme Immunoassay

The data depicted in Table 7 and FIG. 4 were obtained using the protocoldescribed in Example 1 for detecting PCP in a hair extract sample byhomogeneous immunoassay using PCP antibody, G6PDH-PCP conjugate, and ahair extract with PCP calibrator and PCP controls.

TABLE 7 PCP Conc. (ng/10 mg hair) ΔmAU 0 0.4 1.5 29.8 3 47.6 6 90.3

Example 10: Calibration and Determination of Methamphetamine in HairExtract by Homogeneous Enzyme Immunoassay

The data depicted in Table 8 and FIG. 5 were obtained using the protocoldescribed in Example 1 for detecting methamphetamine in a hair extractsample by homogeneous immunoassay using methamphetamine antibody,G6PDH-methamphetamine conjugate, and a hair extract with methamphetaminecalibrator and methamphetamine controls.

TABLE 8 Methamphetamine Conc. (ng/10 mg hair) ΔmAU 0 1.02 1 27.4 2 56.74 113.6

Other Embodiments

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for determining the presence or absence of an analyte ofinterest in a keratinized structure sample comprising: (a) combining inan aqueous medium: i. a conjugate comprising an enzyme and the analyteof interest; ii. an antibody specific for the analyte of interest; andiii. the keratinized structure sample; and (b) determining if theanalyte of interest is present or absent in the keratinized structuresample.
 2. The method of claim 1, wherein the enzyme is selected fromthe group consisting of glucose-6-phosphate dehydrogenase (G6PDH),alcohol dehydrogenase, glutamic dehydrogenase, malic dehydrogenase,isocitric dehydrogenase, α-glycerol phosphate dehydrogenase, lacticdehydrogenase, glyceraldehydes-3-phosphate dehydrogenase, gluthathionereductase, quinine reductase, nitrate reductase, and glutamicdehydrogenase.
 3. The method of claim 1, wherein the enzyme isglucose-6-phosphate dehydrogenase (G6PDH).
 4. The method of claim 3,wherein the G6PDH is covalently linked to the analyte of interest. 5.The method of claim 1, wherein the antibody specific for the analyte ofinterest is specific for an analyte selected from the group consistingof cocaine, benzoylecgonine, cocaethylene, norcocaine, PCP, amphetamine,methamphetamine, cannabinoids, THC, carboxy-THC, heroin,benzodiazepines, methadone, codeine, morphine, 6-monoacetylmorphine(MAM), oxycodone, 3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA).
 6. The method of claim 1,wherein the antibody is detectably labeled.
 7. The method of claim 3,wherein the G6PDH is obtained from a natural source.
 8. The method ofclaim 3, wherein the G6PDH is a recombinant enzyme.
 9. The method ofclaim 3, wherein the aqueous medium further comprises: (a) an enzymesubstrate for G6PDH; and (b) a coenzyme for G6PDH.
 10. The method ofclaim 9, further comprising: detecting a change in the enzymaticactivity of the enzyme-analyte conjugate, wherein the change inenzymatic activity is related to the amount of the analyte of interestpresent in the sample.
 11. The method of claim 1, wherein the antibodyis present at a concentration from about 0.4 μg/mL to about 1.0 μg/mL.12. The method of claim 1, wherein the conjugate comprising an enzymeand the analyte of interest is present at a concentration from about 0.4μg/mL to about 0.5 μg/mL.
 13. The method of claim 1, wherein thedetermining the presence or absence of the analyte of interest isdetermined at a temperature of about 4° C. to about 45° C.
 14. Themethod of claim 1, wherein the determining the presence or absence ofthe analyte of interest is determined at a temperature of about 30° C.to about 40° C.
 15. The method of claim 1, wherein the determining thepresence or absence of the analyte of interest is determined at atemperature of about 37° C.
 16. The method of claim 1, wherein theaqueous medium has a pH range of about 4.0 to about 11.0.
 17. The methodof claim 1, wherein the aqueous medium has a pH of about 7.0.
 18. Themethod of claim 1, wherein the aqueous medium contains less than about20% of a polar solvent.
 19. The method of claim 1, wherein the aqueousmedium contains less than about 20% of N,N-Dimethylformamide (DMF),acetonitrile, dimethylsulfoxide (DMSO), methanol, or a combinationthereof.
 20. The method of claim 1, wherein the analyte of interest isselected from the group consisting of a drug of abuse, a toxic chemical,an environmental chemical, a petroleum product, a natural product, anorganic compound, a nutrient, a prescription and over-the-countermedication, or a metabolite, derivative, or breakdown product of any ofthe foregoing.
 21. The method of claim 20, wherein the analyte ofinterest is a drug of abuse or metabolite thereof.
 22. The method ofclaim 21, wherein the drug of abuse or metabolite thereof is selectedfrom the group consisting of cocaine, benzoylecgonine, cocaethylene,norocaine, PCP, amphetamine, methamphetamines, cannabinoids, THC,carboxy-THC, nicotine, cotinine, benzodiazepines, heroin, codeine,morphine, 6-monoacetylmorphine (MAM), oxycodone,3,4-methylenedioxyamphetamine (MDA), and3,4-methylenedioxymethamphetamine (MDMA).
 23. The method of claim 1,wherein the analyte of interest is selected from the group consisting ofopioids, amphetamines, NSAIDS, steroids, cannabinoids, benzodiazepines,barbiturates, tricyclics, nicotine, and ephedrines, or metabolites,derivatives, or breakdown products of any of the foregoing.
 24. A methodfor determining the presence or absence of an analyte of interest in akeratinized structure sample comprising: (a) combining in an aqueousmedium: i. an enzyme-analyte conjugate comprising glucose-6-phosphatedehydrogenase (G6PDH) covalently linked to the analyte of interest; ii.an antibody specific for the analyte of interest; iii. a keratinizedstructure sample; iv. an enzyme substrate for G6PDH; and v. a coenzymefor G6PDH; and (b) determining if the analyte of interest is present orabsent in the keratinized structure sample.
 25. A method for determiningthe presence or absence of an analyte of interest in a keratinizedstructure sample comprising: (a) combining in an aqueous medium: i. anenzyme-analyte conjugate comprising glucose-6-phosphate dehydrogenase(G6PDH) covalently linked to the analyte of interest; ii. an antibodyspecific for the analyte of interest; iii. a keratinized structuresample; iv. an enzyme substrate for G6PDH; and v. a coenzyme for G6PDH;and (b) determining if the analyte of interest is present or absent inthe keratinized structure sample, wherein the analyte of interest is adrug of abuse or metabolite thereof.
 26. The method of claim 1, whereinthe keratinized structure sample is a hair sample.
 27. The method ofclaim 1, wherein the keratinized structure sample is a nail sample. 28.The method of claim 1, further comprising extracting the analyte ofinterest from the keratinized structure using a buffer.
 29. The methodof claim 28, wherein the buffer is a phosphate buffer.
 30. The method ofclaim 25, further comprising extracting the analyte of interest from thekeratinized structure using a buffer.